CN108707201A - A kind of Arab's gala oligomeric polysaccharide and its preparation and application - Google Patents
A kind of Arab's gala oligomeric polysaccharide and its preparation and application Download PDFInfo
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- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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Abstract
The invention discloses a kind of Arabic gala oligomeric polysaccharide and its preparations and application.Arab's gala oligomeric polysaccharide is made of the polysaccharide that weight percentage is 99% or more, and the polysaccharide is made of the galactolipin of the arabinose and weight percentage 97.0% of weight percentage 3.0%；Its weight average molecular weight is 25KDa.Preparation method is included in the benign environment of pH value 6.0-8.0 obtains weight average molecular weight 25KDa Arab gala oligomeric polysaccharide using ultrasonic wave added free radical orientation degradation arabogalactan, and degradation time is substantially reduced, solve the problems, such as that polysaccharide cannot be degraded to a certain fixed value molecular weight by the prior art.Arab's gala oligomeric polysaccharide bioactivity is remarkably reinforced, and has stronger antioxidant radical activity, can be used directly as antioxidant or be used to prepare antioxidant, can be used for food additives, health products and field of medicaments.
Description
Technical field
The invention belongs to polysaccharide technical fields, and in particular to a kind of Arab's gala oligomeric polysaccharide and its preparation and application.
Background technology
Arabogalactan (AG) is a kind of long, height branch hemicellulose being made of arabinose and galactolipin
Element.Its main chain is galactan, and branch is mainly arabinose side chains, and by β -1,3 keys or β -1,6 keys connect with gala sugar chain
It connects.Largely contain this sugar in the xylem of coniferous tree, up to 25% especially in larch (Larix).Recent study is sent out
Existing, arabogalactan is a kind of important bioactive substance, has immunological regulation, antitumor, adjusting function of intestinal canal etc.
Active function.
Foreign countries are more early to the research starting of arabogalactan, are food by U.S. FDA authentication approval in 2002
Product additive.But the chain molecular weight of arabogalactan is larger, activity is low, needs to improve its activity by strand of degrading,
It could enter and play bioactivity in organism.Foreign countries are studies have found that arabogalactan can be used as a kind of drug load
Body, but as a kind of functional drug, activity is not strong.Some researches show that orientation degradation of polysaccharide can improve the life of polysaccharide
Object activity, it is more to the Study on degradation of Pectic polysaccharides, and not yet appear in the newspapers to arabogalactan.It is existing to other
The research of polysaccharide degradation, discloses a kind of preparation method of Hericium erinaceus active polysaccharide in Chinese patent application CN106478829 A,
It applies the Hericium erinaceus property polysaccharide degraded of supercritical ultrasonics technology, and method is easy to operate, can by the molecular weight of Hericium erinaceus Polysaccharides from
2390000 Da are degraded to 200,000 Da-50, ten thousand Da, and this polysaccharide viscosity is small, and dissolubility is good, and ion vitro immunization is good.Chinese patent
A kind of method preparing small-molecular-weight functional polysaccharide and oligosaccharides using polysaccharides is disclosed in ZL201310085095.7, it should
The ethyl alcohol of certain volume, this method profit are added in research using supercritical ultrasonics technology and degradation agent degradation polysaccharides and during degradation
Keep degradation more abundant with the ethyl alcohol constantly added, and makes polysaccharide molecular weight range relative decrease.Chinese patent
A kind of free radical degradation method of polysaccharide is disclosed in ZL03112409.7, using organic acid, H2O2With ascorbate degradation system
It degrades to polysaccharide, which can control oxygen free radical reaction activity and reduce its toxicity；It should be research shows that free radical drops
Solution reaction condition is mild, and production cost is relatively low, and production technology is feasible, and can control the molecular weight of polysaccharide in 2KDa-30KDa
Range.Disclosed in 106046187 A of Chinese patent application CN it is a kind of have improve immunocompetent sunset abelmoschus stem or bark leaf polyose
Free radical cracking product and preparation method thereof, the research use H2O2- Vc systems are degraded, and it is more to obtain sunset abelmoschus root cauline leaf for purifying
The free radical cracking product of sugar.It is difficult using free radical method degradation of polysaccharide merely although free radical method degradation of polysaccharide effect is preferable
With control, the amount of free radical is excessive, and the polysaccharide molecule chain cut at random also accordingly increases, irregularities；And polysaccharide molecule
Amount is degraded too severe, causes active polysaccharide that cannot fully play；And the amount of free radical is very few, and be difficult to attack polysaccharide
In the target site of chain, the polysaccharide of required molecular weight cannot be obtained.Therefore, independent free radical cracking polysaccharide, it is also difficult to reach institute
The effect needed.Existing Degradation of Polysaccharides has not been achievable at present obtains the sugar of fixed value molecular weight by polysaccharide orientation degradation.
Invention content
The object of the present invention is to provide a kind of Arabic gala oligomeric polysaccharides, and with bioactivity, and activity is compared with me
Primary galactan is remarkably reinforced.
Another object of the present invention is to provide the preparation method of the Arabic gala oligomeric polysaccharide, using ultrasonic wave added freedom
Base orients degradation arabogalactan in the benign environment of pH value 6.0-8.0 and obtains small-molecular-weight oligomeric polysaccharide, can be significantly
Shorten degradation time, the Arabic gala oligomeric polysaccharide (Oligo-AG) of certain molecular weight, and this segment molecule amount are obtained after degradation
Active polysaccharide be remarkably reinforced.
The present invention also provides the applications of the Arabic gala oligomeric polysaccharide, make with stronger oxygen radical removing
With, can be used as antioxidant use, it can also be used to prepare antioxidant.
The technical solution adopted by the present invention to solve the technical problems is：
A kind of Arab's gala oligomeric polysaccharide, the polysaccharide for being 99% or more by weight percentage form, the polysaccharide by
The arabinose of weight percentage 3.0% is formed with the galactolipin of weight percentage 97.0%；Arabic gala is oligomeric more
The weight average molecular weight of sugar is 25KDa.
The preparation method of Arab's gala oligomeric polysaccharide, including step：
(1) it degrades：Arabogalactan aqueous solution is obtained by arabogalactan is soluble in water, and 0.1mmol/ is added
L-1.6mmol/L copper acetates (Cu (Ac)2) aqueous solution, with 0.1mol/L-2.5mol/L NaOH aqueous solution tune pH to 6.0-8.0；
It is first preheated at 40 DEG C -80 DEG C, volume fraction 1%-6%H is added2O2Aqueous solution, mixing；In 40 DEG C of -80 DEG C of heating in ultrasonic wave
It is stirred to react 40min-80min；Then mass percentage concentration 0.1%-0.9%NaHSO is added3Aqueous solution terminates reaction；Centrifugation
It goes to precipitate, obtains supernatant；
Using H2O2Before ultrasonic degradation, addition 0.1mmol/L-1.6mmol/L acetic acid copper liquors, which mainly rise, urges
The effect of agent, hydrogen peroxide can quickly generate OH, attack sugar chain rapidly under the catalysis of transition metal, capture and connect with carbon phase
H atom, generate hydroxyalkyl radical, to greatly improve reaction efficiency.Degradation is sharp in the benign environment of pH value 6.0-8.0
H is assisted with ultrasonic wave2O2, can realize the orientation degradation of arabogalactan, then use mass percentage concentration 0.1%-
0.9%NaHSO3It is to allow degradation reaction no longer to carry out, polysaccharide is prevented to be excessively degraded, cannot get mesh that aqueous solution, which terminates reaction,
Mark product.
(2) it dialyses：By the supernatant obtained by step (1) be 4000Da-8000Da with aperture bag filter in deionized water
Middle dialysis, collects the extracting solution after dialysis, and vacuum freeze drying obtains Thick many candies；
(3) it purifies：The Thick many candies that step (2) obtains are obtained into Thick many candies aqueous solution with deionized water dissolving, through propylene Portugal
Polysaccharide gel filtration chromatography is purified, and the eluent containing polysaccharide is detected with phend-sulphuric acid collected by gel permeation chromatography
It is oligomeric that polysaccharide peak, the maximum eluent containing polysaccharide of the absorbance of collection is concentrated, dialysis and freeze-drying obtain Arabic gala
Polysaccharide.
In order to reach better invention effect, preferably：
In step (1), commercial product can be used in the arabogalactan, and existing preparation method can also be used and be prepared into
It arrives.Such as the arabogalactan that water extraction and alcohol precipitation method obtains can be used.
The arabogalactan extracted from Larch may be used in the arabogalactan.The larch
Northeast Larch can be used in wood.
The extracting method of the arabogalactan includes：By larch sawdust at 80 DEG C -98 DEG C according to solid-liquid ratio 1:
10-1:30 (m/V, g/mL) hot water extraction 40min-80min；First-time filtrate is filtered to obtain, filter residue is at 80 DEG C -98 DEG C according to solid-liquid ratio
1:10-1:30 (m/V, g/mL) hot water extraction 40min-80min, filter to obtain secondary filtrate, collect whole filtrates, 40 DEG C -80 DEG C
It is concentrated under reduced pressure；The ethanol water that concentrate is added and accounts for 3 times of -5 times of amounts of the volume of the concentrated liquid, volume fraction is 90%-95% is in 2
DEG C -6 DEG C stand overnight；Centrifugation collects precipitation through being dried to obtain arabogalactan.Wherein, the condition of centrifugation may be selected to be
5000r/min-6000r/min centrifuges 5min-10min.
The ratio between the weight of arabogalactan and the volume of water are 0.1g- in the arabogalactan aqueous solution
2.0g：100ml-200ml.
The acetic acid copper liquor is mainly used as catalyst, the not stringent limitation of dosage, the general vinegar in terms of copper acetate
The mass ratio of sour copper and arabogalactan is 1:100-120.
The H2O2The dosage of aqueous solution is with H2O2Meter, H2O2Mass ratio with arabogalactan is 5-10:100.
In step (2), the time dialysed in deionized water is 18h-32h.
In step (3), a concentration of 5mg/mL-20mg/mL of the Thick many candies aqueous solution, further preferably 5mg/mL-
8mg/mL.When chromatography, the flow velocity of the Thick many candies aqueous solution is 0.5ml/min-1.2ml/min.
The condition of the Sephacryl filtration chromatography is：Eluent be 0.05mol/L phosphate buffers and
The volume ratio of the NaCl aqueous solutions of 0.15mol/L, flow velocity 0.5ml/min, wherein phosphate buffer and NaCl aqueous solutions is 2-
3:1。
The Sephacryl selects commercial product, such as commercially available SephacrylTmS-100HR etc..
The preparation method of the phosphate buffer generally can refer to version in 2005 according to method generally in the art《In
State's pharmacopeia》.
In step (3), the dialysis uses aperture to dialyse in deionized water for the bag filter of 4000Da-8000Da.
The absorbance is the absorbance of 510nm.
Arab's gala oligomeric polysaccharide has the ability of scavenging activated oxygen, is measured in OH free radical scavenging activities
In, under the premise of same concentrations, the OH free radical scavenging activities of Arabic gala oligomeric polysaccharide are much larger than arabinogalactan
The OH free radical scavenging activities of sugar；Show that Arabic gala oligomeric polysaccharide of the invention has stronger antioxidant radical activity, it can
To use directly as antioxidant or be used to prepare antioxidant, it can be used for food additives, health products and field of medicaments.
Compared with prior art, the invention has the advantages that：
Arab's gala oligomeric polysaccharide weight average molecular weight of the invention can be fixed to 25KDa, and the list of its saccharide portion
Sugar group becomes arabinose and galactolipin, and the ratio between weight percentage of arabinose and galactolipin is 3.0:97.0, this is just
Practical application for natural polysaccharide product provides technical support, has the foreground of being widely applied.
Arab's gala oligomeric polysaccharide of the invention is compared with arabogalactan, and bioactivity is remarkably reinforced, and hydroxyl is free
Scavenging activity is also remarkably reinforced, and has stronger antioxidant radical activity, can use or be used for directly as antioxidant
Antioxidant is prepared, food additives, health products and field of medicaments are can be used for.
The method of the present invention is Arabic using ultrasonic wave auxiliary free radical orientation degradation in the benign environment of pH value 6.0-8.0
Galactan obtains small-molecular-weight oligomeric polysaccharide, can not only greatly shorten degradation time, but also can also be substantially reduced polysaccharide point
Son amount, is a kind of inexpensive and environment-friendly high-efficiency biodegrading process.
Arabogalactan can be oriented and be degraded to fixed value molecular weight and find this segment molecule amount by the method for the present invention
Oligomeric polysaccharide activity be remarkably reinforced, solve the problems, such as that polysaccharide cannot be degraded to a certain fixed value molecular weight by the prior art.
Description of the drawings
Fig. 1 is that Thick many candies pass through the eluent containing polysaccharide collected through Sephacryl filtration chromatography in embodiment 1
In the absorbance curve figure of 510nm.
Fig. 2 is the result figure of arabogalactan absolute molecular quality after degrading in embodiment 4, wherein abscissa is
Appearance time (min), ordinate are the corresponding relative value of instrument.
Fig. 3 is that the GC-MS of monosaccharide standard in embodiment 5 analyzes chromatogram, wherein Rham is rhamnose, and Fuc is rock algae
Sugar, Ara are arabinose, and Xyl is xylose, and Man is mannose, and Glc is glucose, and Gal is galactolipin.
The GC-MS that Fig. 4 is AG in embodiment 5 analyzes chromatogram.
The GC-MS that Fig. 5 is Oligo-AG in embodiment 5 analyzes chromatogram.
Fig. 6 is to compare OH elimination effects before and after the arabogalactan polysaccharide of various concentration in embodiment 7 is degraded
Figure, abscissa are polysaccharide concentration (mg/mL), and ordinate is OH clearance rates (%).
Fig. 7 is the biological evaluation figure of arabogalactan in embodiment 8.
Fig. 8 is the biological evaluation figure of Arabic gala oligomeric polysaccharide in embodiment 8.
Specific implementation mode
In the following with reference to the drawings and specific embodiments, the present invention is further explained.It should be understood that these embodiments are merely to illustrate
The present invention rather than limit the scope of the invention.In addition, it should also be understood that, after reading the content that the present invention lectures, ability
Field technique personnel can make the present invention various changes and modification, and such equivalent forms also belong to the application appended claims
Book limited range.
Embodiment 1
The extraction of arabogalactan：A certain amount of northeast larch sawdust is weighed, at 98 DEG C according to solid-liquid ratio 1:10
(m/V, g/mL) hot water extraction 80min；First-time filtrate is filtered to obtain, filtered filtration residue carries out second extraction:At 98 DEG C according to feed liquid
Than 1:10 (m/V, g/mL) hot water extraction 80min, filter to obtain secondary filtrate；Whole filtrates obtained by collection extraction twice, 40 DEG C
It is concentrated under reduced pressure；It is added in concentrate and accounts for 5 times of amounts of the volume of the concentrated liquid, the ethanol water that volume fraction is 95% is stood in 6 DEG C
Overnight；Centrifugation, 5000r/min centrifuge 10min；It collects precipitation and vacuum dried obtains arabogalactan.
The preparation of Arabic gala oligomeric polysaccharide：
(1) it degrades：By the arabogalactan of extraction, 2.0g is taken to be dissolved in 100ml distilled water, dissolved by heating；It is added
68.75mL、1.6mmol/L Cu(Ac)2Aqueous solution, with 2.5mol/L NaOH aqueous solution tune pH to 8.0；In 40 DEG C of ultrasonic instruments
It is middle first to preheat, 1.16mL, volume fraction 6%H is added2O2Aqueous solution, mixing；Open ultrasonic wave, 40 DEG C of heating stirring reactions
80min；Mass percentage concentration 0.9%NaHSO is added3Aqueous solution terminates reaction.Centrifugation goes to precipitate, and obtains supernatant.
(2) it dialyses：The bag filter that supernatant obtained by step (1) is 4000Da with aperture is dialysed in deionized water
32h, the process can remove oligosaccharides, pigment, organic solvent and inorganic salts etc., collect the extracting solution after dialysis, and vacuum refrigeration is dry
It is dry to obtain Thick many candies.
(3) it purifies：The Thick many candies that step (2) obtains are obtained into 5mg/ml Thick many candies aqueous solutions with deionized water dissolving；It will
Thick many candies aqueous solution is through Sephacryl filtration chromatography (SephacrylTmS-100HR it) is further purified, the rule of chromatographic column
Lattice are 2.6cm × 100cm, and the flow velocity of applied sample amount 5ml, Thick many candies aqueous solution are 0.5ml/min, and eluent is 0.05mol/L phosphorus
(volume ratio of wherein phosphate buffer and NaCl aqueous solutions is phthalate buffer (pH7.0)+0.15mol/L NaCl aqueous solutions
2:1), eluent flow rate 0.5ml/min, the collected eluent containing polysaccharide of gel permeation chromatography are detected with phend-sulphuric acid
Polysaccharide peak, the bag filter dialysis and freeze that concentrated, aperture is 4000Da by the maximum eluent containing polysaccharide of the absorbance of collection
It is dry to obtain Arabic gala oligomeric polysaccharide, it is named as Oligo-AG.
Abs values (absorbance) mapping of polysaccharide, such as Fig. 1 are measured according to step (3) phend-sulphuric acid.Fig. 1 highests are collected to inhale
Receive sample (its Abs value is 1) the i.e. maximum eluent containing polysaccharide of absorbance of peak test tube (the 18th pipe), concentrated, dialysis and
The product obtained after freeze-drying is Arabic gala oligomeric polysaccharide, is named as Oligo-AG.
After testing, the weight average molecular weight of Oligo-AG is 25KDa.
Embodiment 2
The extraction of arabogalactan：A certain amount of northeast larch sawdust is weighed, at 80 DEG C according to solid-liquid ratio 1:30
(m/V, g/mL) hot water extraction 40min；First-time filtrate is filtered to obtain, filtered filtration residue carries out second extraction:At 80 DEG C according to feed liquid
Than 1:30 (m/V, g/mL) hot water extraction 40min, filter to obtain secondary filtrate；Whole filtrates obtained by collection extraction twice, 80 DEG C
It is concentrated under reduced pressure；It is added in concentrate and accounts for 3 times of amounts of the volume of the concentrated liquid, the ethanol water that volume fraction is 90% is stood in 2 DEG C
Overnight；Centrifugation, 6000r/min centrifuge 5min；It collects precipitation and vacuum dried obtains arabogalactan.
The preparation of Arabic gala oligomeric polysaccharide：
(1) it degrades：By the arabogalactan of extraction, 0.1g is taken to be dissolved in 100ml distilled water, dissolved by heating；It is added
45.88mL、0.1mmol/L Cu(Ac)2Aqueous solution, with 0.1mol/L NaOH aqueous solution tune pH to 6.0；In 80 DEG C of ultrasonic instruments
It is middle first to preheat, 0.69mL, volume fraction 1%H is added2O2Aqueous solution, mixing；Open ultrasonic wave, 80 DEG C of heating stirring reactions
40min；Mass percentage concentration 0.1%NaHSO is added3Aqueous solution terminates reaction.Centrifugation goes to precipitate, and obtains supernatant.
(2) it dialyses：The bag filter that supernatant obtained by step (1) is 8000Da with aperture is dialysed in deionized water
20h, the process can remove oligosaccharides, pigment, organic solvent and inorganic salts etc., collect the extracting solution after dialysis, and vacuum refrigeration is dry
It is dry to obtain Thick many candies.
(3) it purifies：The Thick many candies that step (2) obtains are obtained into 6mg/ml Thick many candies aqueous solutions with deionized water dissolving；It will
Thick many candies aqueous solution is through Sephacryl filtration chromatography (SephacrylTmS-100HR it) is further purified, the rule of chromatographic column
Lattice are 2.6cm × 100cm, and the flow velocity of applied sample amount 5ml, Thick many candies aqueous solution are 1.2ml/min, and eluent is 0.05mol/L phosphorus
(volume ratio of wherein phosphate buffer and NaCl aqueous solutions is phthalate buffer (pH7.0)+0.15mol/L NaCl aqueous solutions
2:1), eluent flow rate 0.5ml/min, the collected eluent containing polysaccharide of gel permeation chromatography are detected with phend-sulphuric acid
Polysaccharide peak, collection is saturating in the bag filter that the maximum eluent containing polysaccharide of 510nm absorbances is concentrated, aperture is 8000Da
Analysis and freeze-drying obtain Arabic gala oligomeric polysaccharide, are named as Oligo-AG.
After testing, the weight average molecular weight of Oligo-AG is 25KDa.
Embodiment 3
The extraction of arabogalactan：A certain amount of northeast larch sawdust is weighed, at 88 DEG C according to solid-liquid ratio 1:20
(m/V, g/mL) hot water extraction 65min；First-time filtrate is filtered to obtain, filtered filtration residue carries out second extraction:At 88 DEG C according to feed liquid
Than 1:20 (m/V, g/mL) hot water extraction 65min, filter to obtain secondary filtrate；Whole filtrates obtained by collection extraction twice, 65 DEG C
It is concentrated under reduced pressure；It is added in concentrate and accounts for 4 times of amounts of the volume of the concentrated liquid, the ethanol water that volume fraction is 95% is stood in 4 DEG C
Overnight；Centrifugation, 6000r/min centrifuge 6min；It collects precipitation and vacuum dried obtains arabogalactan.
The preparation of Arabic gala oligomeric polysaccharide：
(1) it degrades：By the arabogalactan of extraction, 1.2g is taken to be dissolved in 200ml distilled water, dissolved by heating；Add
60.06mL、1.0mmol/L Cu(Ac)2Aqueous solution, with 1.5mol/L NaOH aqueous solution tune pH to 7.0；In 70 DEG C of ultrasonic instruments
It is middle first to preheat, 2.22mL, volume fraction 3%H is added2O2Aqueous solution, mixing；Open ultrasonic wave, 60 DEG C of heating stirring reactions
60min；Mass percentage concentration 0.6%NaHSO is added3Aqueous solution terminates reaction.Centrifugation goes to precipitate, and obtains supernatant.
(2) it dialyses：The bag filter that supernatant obtained by step (1) is 6000Da with aperture is dialysed in deionized water
18h, the process can remove oligosaccharides, pigment, organic solvent and inorganic salts etc., collect the extracting solution after dialysis, and vacuum refrigeration is dry
It is dry to obtain Thick many candies.
(3) it purifies：The Thick many candies that step (2) obtains are obtained into 8mg/ml Thick many candies aqueous solutions with deionized water dissolving；It will
Thick many candies aqueous solution is through Sephacryl filtration chromatography (SephacrylTmS-100HR it) is further purified, the rule of chromatographic column
Lattice are 2.6cm × 100cm, and the flow velocity of applied sample amount 5ml, Thick many candies aqueous solution are 1.0ml/min, and eluent is 0.05mol/L phosphorus
(volume ratio of wherein phosphate buffer and NaCl aqueous solutions is phthalate buffer (pH7.0)+0.15mol/L NaCl aqueous solutions
3:1), eluent flow rate 0.5ml/min, the collected eluent containing polysaccharide of gel permeation chromatography are detected with phend-sulphuric acid
Polysaccharide peak, collection is saturating in the bag filter that the maximum eluent containing polysaccharide of 510nm absorbances is concentrated, aperture is 6000Da
Analysis and freeze-drying obtain Arabic gala oligomeric polysaccharide, are named as Oligo-AG.
After testing, the weight average molecular weight of Oligo-AG is 25KDa.
Here is the embodiment to Oligo-AG Structural Identifications or performance evaluation：
Embodiment 4：Molecular weight detection
Specific experiment process：Sample size is 200 μ L-300 μ L (i.e. 2-3 times of sample loop volume), data collection 60min, column
Temperature is room temperature.Prepare 0.1mol/L NaNO3Aqueous solution (contains mass percentage concentration 0.02%NaN3) it is used as mobile phase, cross 0.22
μm film, ultrasound degassing 30min.It weighs 3mg samples and is dissolved in 1mL0.1mol/L NaNO3Aqueous solution (contains mass percentage concentration
0.02%NaN3) in, magnetic agitation dissolves 4h, crosses 0.22 μm of film.Sample uses the Thick many candies in embodiment 1, polysaccharide after degradation
Absolute weight average molecular result see Fig. 2.
What Fig. 2 reflected is the result figure of the absolute molecular weight of ultrasonic wave added free radical cracking polysaccharide degradation effect of the present invention,
As shown in Figure 2, ultrasonic wave added free radical cracking method of the present invention enhances the degradation of macromolecule range, can get a large amount of 10KDa
The molecule of molecular weight, and so that the small molecule mass percentage of 7KDa-25KDa is reached 60% and (calculated according to peak area ratio
To).And the activity through the Arabic gala oligomeric polysaccharide that determination of light scattering absolute weight average molecular mass (Mw) is 25KDa significantly increases
Add, this is just that oriented control and the Arabic gala oligomeric polysaccharide of application are laid a good foundation.
Embodiment 5：GC-MS is analyzed.
The hydrolysis of polysaccharide：2mgAG and 2mg Oligo-AG samples are weighed respectively, and the trifluoroacetic acid (TFA) of 2mol/L is added
Aqueous solution, close plug hydrolyze 4h in 120 DEG C of vacuum drying chamber, are cooled to room temperature, and 0.5ml methanol is added and is depressurized at 40 DEG C
It is spin-dried for, 4 times repeatedly (amount that methanol is added when repeatedly every time is 2ml), thoroughly to remove TFA.
The preparation of derivative：Each standard specimen being spin-dried for and sample is open, it is placed in drier, drier bottom, which is placed with, to be added
The culture dish of phosphorus pentoxide.Hermetically drying device, vacuumizes, and is dried overnight.
The derivatization of monosaccharide and simple monosaccharide:Article after drying is separately added into the hydroxylamine hydrochloride solution of 1ml, closed and fill
Divide concussion, is placed in 90 DEG C of vacuum drying chambers and aoxidizes 30 minutes.It is cooled to room temperature, 0.2ml 1- methylimidazoles and 1ml is added
Acetic anhydride fully shaking is uniformly mixed.It is closed, it is placed in 90 DEG C of vacuum drying chambers and reacts 30 minutes.It is cooled to room temperature to form acetyl
The derivative of change.
The water of chloroform (chloroform) and 1ml of 1ml is added in the derivative of acetylation.Fully shaking.Stand several points
Clock waits being layered, and removes upper strata aqueous phase.The water of 1ml is added, fully shaking is stood, and removes water phase.5 times repeatedly.Obtained chloroform
Layer is added appropriate anhydrous sodium sulfate and removes moisture removal.Chloroform layer is drawn with band needle injection, the organic system filter membrane of 0.45nm is crossed, is placed in
Wait for that GC is detected in sample injection bottle.
Chromatographiccondition HP-Innowax capillary columns (250 μ m 30m), 0.20 μm of thickness of liquid film, chromatographic column temperature 190
DEG C, 280 DEG C of temperature of vaporization chamber, split ratio 10:1, fid detector detects 250 DEG C of temperature, 1 μ L of sample size；Carrier gas is helium, gas
Body flow velocity 1mL/min.
The GC-MS of AG and Oligo-AG is analyzed as follows：
The GC-MS analysis chromatograms of monosaccharide standard are as shown in figure 3, the GC-MS analysis chromatograms of AG are as schemed in embodiment 1
Shown in 4, the GC-MS analysis chromatograms of Oligo-AG are as shown in Figure 5 in embodiment 1.
AG is made of the polysaccharide that weight percentage is 99% or more as shown in Figure 4, and the monosaccharide of saccharide portion is by me
Uncle's sugar is formed with galactolipin, and the ratio between arabinose (Ara) and the weight percentage of galactolipin (Gal) are 6.0:94.0；Explanation
AG is the polysaccharide using galactolipin as main chain, and containing branch.
Corresponding monosaccharide standard, the polysaccharide group that the Oligo-AG of embodiment 1,2 or 3 is 99% or more by weight percentage
At the monosaccharide of saccharide portion is made of galactolipin and arabinose, is mainly made of galactolipin, and arabinose only accounts for seldom
Amount, and the ratio between weight percentage of arabinose and galactolipin is 3.0:97.0, illustrate what AG was obtained after degradation
Oligo-AG is a kind of main group of polysaccharide for becoming galactolipin, and the branch of polysaccharide chain is few.
Embodiment 6：Comparison of the measurement and AG and Oligo-AG of Scavenging action to hydroxyl free radical to the clearance rate of OH：
Principle is：It is reacted using Fenton and generates hydroxy radical：H2O2+Fe2+=OH+H2O+Fe3+In the reaction system
Salicylic acid is added, the hydroxy radical and salicylism reaction that Fenton reactions generate, being created on has the coloured of absorption maximum at 510nm
Substance.The content of hydroxy radical is indicated with the light absorption value.
It is as follows：1mL 9mmol/L FeSO are taken respectively4Aqueous solution, 1mL 9mmol/L salicylic acids ethanol solution,
1mL 8.8mmol/L H2O2Aqueous solution, is added each 1mL of prepared sample, mixing, and 37 DEG C of water-bath 30min are made empty with distilled water
In vain, the absorbance of each solution is measured at 510nm.Experiment sets 3 groups：Sample sets A1, control group A0, background group A2.Clearance rate meter
Calculation method：
In formula：A0To be not added with the absorbance of sample solution；A1For the absorbance of sample solution is added；A2To be not added with color developing agent
When sample itself absorbance.
0.1mg/mL, 0.3mg/mL, 0.5mg/mL, 0.7mg/mL, 1.0mg/mL, 2.0mg/mL, 3.0mg/ are prepared respectively
The AG aqueous solutions of mL, 4.0mg/mL and the Oligo-AG aqueous solutions obtained through embodiment 1-3, are tested as sample, are compared
Clearance rate of the sample to OH.Measurement result is shown in Fig. 6.
As seen from Figure 6, arabogalactan, Arabic gala oligomeric polysaccharide have hydroxy radical certain clear
Except effect, and within the scope of experimental concentration, the scavenging effect to hydroxy radical is enhanced with the increase of polysaccharide concentration
's.When polysaccharide mass concentration reaches 0.5mg/mL, AG, Oligo-AG are respectively 37.1%, 50.3% to the clearance rate of OH.Separately
Outside by can be calculated, the IC of Oligo-AG50=0.497mg/mL, and the IC of AG50=2.058mg/mL.In same concentrations
Under the premise of, the OH free radical scavenging activities of Arabic gala oligomeric polysaccharide are clear much larger than the OH free radicals of arabogalactan
Except rate.The result shows that Oligo-AG is stronger to the scavenging effect of hydroxyl radical free radical, and it is apparently higher than AG, it can be directly as anti-
Oxidant uses or is used to prepare antioxidant, can be used for food additives, health products and field of medicaments.
Embodiment 7：To the biological evaluation of AG and Oligo-AG
The polysaccharide that embodiment 1 is obtained, with the 3000 system measurement differences of BIAcore point of surface plasma resonance (SPR)
The bonding behavior of the galactan and galectin-3 (Gal-3) of son amount.First, by using 1- (3- dimethylaminos third
Base) Gal-3 is fixed on CM5 chips by -3- ethyl-carbodiimide hydrochlorides (EDC)/n-hydroxysuccinimide (NHS) amine coupling
On.The Arabic gala oligomeric polysaccharide sample of different diluted concentrations is loaded with the flow of 30 μ l/min, and HBS-EP buffer solutions pass through
Sensor surface is detached.Sensor surface is complete after dissociation in 3 minutes by injecting the lactose of 150mM (mmol/L)
Regenerated surface.
Can be obtained by Fig. 7 and Fig. 8, AG-II (arabogalactan extracted from larch sawdust be two types I
Primary galactan) bond strength kd/ka values be 2.58 μM (μm ol/L)；The bond strength kd/ka values of Oligo-AG are
64.4nM(nmol/L).Compared with arabogalactan, the weight average molecular weight that ultrasonic wave added free radical method of the present invention obtains is
The binding force of the sample Oligo-AG and Gal-3 of 25KDa are remarkably reinforced, and show that the bioactivity of Oligo-AG is notable relative to AG
Enhancing.
The variation of parameter has no effect on the preparation of Arabic gala oligomeric polysaccharide, therefore the present invention in preparation method of the present invention
The preparation of Arabic gala oligomeric polysaccharide can be achieved in the combination of arbitrary parameter in preparation method.Details are not described herein.
Claims (10)
1. a kind of Arab's gala oligomeric polysaccharide, which is characterized in that the polysaccharide for being 99% or more by weight percentage forms, institute
Polysaccharide is stated to be made of the galactolipin of the arabinose and weight percentage 97.0% of weight percentage 3.0%；Arabic half
The weight average molecular weight of newborn oligomeric polysaccharide is 25KDa.
2. the preparation method of Arab's gala oligomeric polysaccharide according to claim 1, which is characterized in that including step：
(1) it degrades：Arabogalactan aqueous solution is obtained by arabogalactan is soluble in water, and 0.1mmol/L- is added
1.6mmol/L acetic acid copper liquors, with 0.1mol/L-2.5mol/L NaOH aqueous solution tune pH to 6.0-8.0；At 40 DEG C -80 DEG C
It first preheats, volume fraction 1%-6%H is added2O2Aqueous solution, mixing；It is reacted in 40 DEG C of -80 DEG C of heating stirrings in ultrasonic wave
40min-80min；Then mass percentage concentration 0.1%-0.9%NaHSO is added3Aqueous solution terminates reaction；Centrifugation goes to precipitate, and obtains
To supernatant；
(2) it dialyses：By the supernatant obtained by step (1) with aperture be 4000Da-8000Da bag filter it is saturating in deionized water
Analysis, collects the extracting solution after dialysis, and vacuum freeze drying obtains Thick many candies；
(3) it purifies：The Thick many candies that step (2) obtains are obtained into Thick many candies aqueous solution with deionized water dissolving, through propylene glucan
Gel permeation chromatography is purified, and the collected eluent containing polysaccharide of gel permeation chromatography detects polysaccharide with phend-sulphuric acid
Peak, the maximum eluent containing polysaccharide of the absorbance of collection is concentrated, dialysis and freeze-drying obtain Arabic gala oligomeric polysaccharide.
3. preparation method according to claim 2, which is characterized in that in step (1), the arabogalactan is to adopt
The arabogalactan obtained with water extraction and alcohol precipitation method.
4. preparation method according to claim 2 or 3, which is characterized in that in step (1), the arabogalactan
It is the arabogalactan extracted from Larch.
5. preparation method according to claim 4, which is characterized in that the extracting method packet of the arabogalactan
It includes：By larch sawdust at 80 DEG C -98 DEG C according to solid-liquid ratio 1:10-1:30 hot water extraction 40min-80min；It filters once to filter
Liquid, filter residue is at 80 DEG C -98 DEG C according to solid-liquid ratio 1:10-1:30 hot water extraction 40min-80min filter to obtain secondary filtrate, collect
Whole filtrates, 40 DEG C of -80 DEG C of reduced pressures；Concentrate addition accounts for the volume of the concentrated liquid 3 times of -5 times of amounts, volume fraction 90%-
95% ethanol water is stood overnight in 2 DEG C -6 DEG C；Centrifugation collects precipitation through being dried to obtain arabogalactan；Feed liquid
The unit of ratio is g/mL.
6. preparation method according to claim 2, which is characterized in that in step (1), the arabogalactan is water-soluble
The ratio between the weight of arabogalactan and the volume of water are 0.1g-2.0g in liquid：100ml-200ml.
7. preparation method according to claim 2, which is characterized in that described to dialyse in deionized water in step (2)
Time is 18h-32h.
8. preparation method according to claim 2, which is characterized in that in step (3), the concentration of the Thick many candies aqueous solution
For 5mg/mL-20mg/mL, flow velocity 0.5ml/min-1.2ml/min.
9. preparation method according to claim 2, which is characterized in that in step (3), the Sephacryl filtering
The condition of chromatography is：Eluent is the NaCl aqueous solutions of 0.05mol/L phosphate buffers and 0.15mol/L, flow velocity 0.5ml/
The volume ratio of min, wherein phosphate buffer and NaCl aqueous solutions is 2-3:1.
10. Arab's gala oligomeric polysaccharide according to claim 1 as antioxidant or is being used to prepare antioxygen
Application in agent.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110734503A (en) * | 2019-11-22 | 2020-01-31 | 浙江省林业科学研究院 | Arab galactooligosaccharides, and preparation and application thereof |
| CN112979837A (en) * | 2021-03-04 | 2021-06-18 | 西北大学 | Preparation method and application of red date arabinogalactan |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101143904A (en) * | 2007-04-17 | 2008-03-19 | 东北林业大学 | Method for preparing high-purity arabinogalactan |
| CN104725520A (en) * | 2015-03-13 | 2015-06-24 | 凤庆县裕泽谷核桃专业合作社 | Diaphragma juglandis acidic polysaccharose and preparation and application thereof |
| CN104961841A (en) * | 2015-07-28 | 2015-10-07 | 东北林业大学 | Method for extracting larch arabinogalactan |
-
2018
- 2018-05-03 CN CN201810413040.7A patent/CN108707201B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101143904A (en) * | 2007-04-17 | 2008-03-19 | 东北林业大学 | Method for preparing high-purity arabinogalactan |
| CN104725520A (en) * | 2015-03-13 | 2015-06-24 | 凤庆县裕泽谷核桃专业合作社 | Diaphragma juglandis acidic polysaccharose and preparation and application thereof |
| CN104961841A (en) * | 2015-07-28 | 2015-10-07 | 东北林业大学 | Method for extracting larch arabinogalactan |
Non-Patent Citations (1)
| Title |
|---|
| 郭怡帆等: ""超声降解落叶松中阿拉伯半乳聚糖及其活性的研究"", 《中国林副特产》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110734503A (en) * | 2019-11-22 | 2020-01-31 | 浙江省林业科学研究院 | Arab galactooligosaccharides, and preparation and application thereof |
| CN110734503B (en) * | 2019-11-22 | 2021-12-21 | 浙江省林业科学研究院 | Arab galacto-oligosaccharide and preparation and application thereof |
| CN112979837A (en) * | 2021-03-04 | 2021-06-18 | 西北大学 | Preparation method and application of red date arabinogalactan |
| CN112979837B (en) * | 2021-03-04 | 2022-02-18 | 西北大学 | Preparation method and application of dog head jujube arabinogalactan |
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